Researchers have invested considerable effort attempting to identify the pharmacogenetic basis of idiosyncratic adverse drug reactions, particularly hypersensitivity reactions. There is clear evidence for pharmacogenetic influence on susceptibility to hypersensitivity reactions. One such pharmacogenetic influence is genetic polymorphism. Genetic polymorphisms involve the regular and simultaneous existence in the same population of two or more discontinuous variants or genotypes in frequencies that cannot be due to recurrent mutations. Probably, the best known example of genetic polymorphism involves the different human blood groups. Genetic polymorphisms at loci which encode enzymes involved in metabolism of toxic or carcinogenic compounds can have clinical implications in drug metabolism. The pharmacokinetic and pharmacodynamic consequences of the activity of a polymorphic enzyme depend upon whether it mediates metabolism of the parent drug, the metabolites or both, whether parent drug or metabolites or both are active, the overall contribution to clearance from the affected pathway, the potency of the active species, and the patency of competing pathways of elimination. Tucker, Journal Pharmacology, 46, 417-424 (1994). Examples of the various permutations have been illustrated by Gram, et al in Clinical Relevance of Genetic Polymorphisms in Drug Oxidation, (1992).
One group of polymorphic enzymes that has implications in drug metabolism is the glucuronosyltransferases. Glucuronosyltransferases (UGTs) are the enzymes responsible for converting endogenous, toxic or carcinogenic compounds into a more water soluble form so they may be excreted from the body. UGTs are members of the transferase class of enzymes and are characterized by their ability to catalyze reactions that transfer functional groups from one compound to another. During metabolism, UGTs render polarity to xenobiotics and other exogenous compounds by catalyzing the transfer of a glucuronate moiety from uridine diphosphate glucuronate to an acceptor xcex2-D-glucuronoside to form a glucuronide conjugate which can then be secreted into the bile. In addition to catalyzing reactions to detoxify exogenous compounds, UGTs catalyze reactions that impart water solubility to endogenous compounds. For example, UGTs catalyze a reaction in the liver whereby bilirubin is rendered water soluble by conjugation to form bilirubin bisglucuronide, most of which is excreted in the bile.
Higher than normal levels of bilirubin in the blood can be caused by one such genetic polymorphism, Gilbert""s Syndrome. Gilbert""s Syndrome (GS) is a benign unconjugated hyperbilirubinemia characterized by the presence of higher than normal concentrations of bilirubin in the blood. The higher than normal levels of bilirubin in the blood often result in episodes of mild intermittent jaundice. Gilbert""s syndrome hyperbilirubinemia occurs in the absence of structural liver disease and overt hemolysis. It is part of a spectrum of familial unconjugated hyperbilirubinemias including the more severe Crigler-Najjar (CN) Syndromes (types 1 and 2). GS is the most common inherited disorder of hepatic bilirubin metabolism occurring in 2-12% of the population and is often detected in adulthood through routine screening blood tests or the fasting associated with surgery or illness which unmasks the hyperbilirubinemia. The most consistent feature in GS is a deficiency in bilirubin glucuronidation but altered metabolism of drugs has also been reported. Altered rates of bilirubin production, hepatic heme production and altered hepatic uptake of bilirubin have been reported in some GS patients. Due to the benign nature of the syndrome and its prevalence in the population it may be more appropriate to consider GS as a normal genetic variant exhibiting reduced bilirubin glucuronidation capacity (which in certain situations such as fasting, illness or administration of drugs) could precipitate jaundice.
In addition to bilirubin metabolism, the glucuronidation detoxification pathway is also responsible for detoxifying many other compounds. For example, two promising antitumor agents, Irinotecan (CPT-11) and TAS-103, are detoxified in the liver by the same pathway. While Irinotecan has been recently approved for repeats use in patients with metastatic colorectal cancer, TAS-103 is currently undergoing Phase I clinical trials in the United States. The active metabolite of Irinotecan, SN-38, and TAS-103 are both glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs) in vivo. The major dose-limiting toxicity of Irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. On the other hand, the major toxicity associated with TAS-103 therapy is leukopenia. In the case of both drugs, glucuronidation is the major detoxification pathway. In patients with GS or CN, where glucuronidation activity is low, increased susceptibility to drug toxicity is a major problem. There remains a need in the art for a method to detect genetic polymorphisms at loci which encode enzymes involved in metabolism of endogenous, toxic or carcinogenic compounds.
The present invention is directed to methods for detecting promoter polymorphisms that correlate with altered gene expression. More specifically, the present invention is directed to methods for determining the presence of genetic polymorphisms within a uridine diphosphate glucuronosyltransferase and or its promoter, and preferably within the uridine diphosphate glucuronosyltransferase I (UGT1A1) promoter. The polymorphisms comprise a variation in the number of thymidine-adenine repeats, TAn, where n=the number of TA repeats. Preferably the polymorphisms comprise the homozygous genotypes [TA]5/[TA]5 and/or [TA]8/[TA]8, as well as the heterozygous genotypes [TA]5/[TA]6, [TA]5/[TA]7, [TA]5/[TA]8, [TA]6/[TA]8, and [TA]7/[TA]8.
The present invention also provides methods for screening individuals for variation in glucuronidation activity by detecting polymorphisms in a UGT promoter by determining the number of TA repeats in that UGT promoter. More specifically, the present invention provides methods for screening individuals for varying glucuronidation activity caused by the presence or absence of alleles [TA]5, [TA]6, [TA]7 and/or [TA]8 in the UGT1A1 promoter.
The present invention provides methods for determining or optimizing drug dosages by detecting polymorphisms in a UGT promoter by determining the number of TA repeats in the UGT promoter. More specifically, the present invention provides methods of determining optimum dosages of drugs based upon the presence or absence of alleles [TA]5, [TA]6, [TA]7 and/or [TA]8, in the UGT1A1 promoter.
In another embodiment, the present invention provides methods for determining or optimizing drug dosages by screening individuals for variation in glucuronidation activity by detecting polymorphisms in a UGT promoter by determining the number of TA repeats in the UGT promoter. More specifically, the present invention provides methods for determining optimum dosages of drugs based upon varying glucuronidation activity caused by the presence or absence of alleles [TA]5, [TA]6, [TA]7 and/or [TA]8, in the UGT1A1 promoter.
The present invention also provides methods for predicting an individual""s sensitivity to xenobiotics by detecting polymorphisms in a UGT promoter by determining the number of TA repeats in that UGT promoter. More specifically, the present invention provides methods for predicting an individual""s sensitivity to xenobiotics caused by the presence or absence of alleles [TA]5, [TA]6, [TA]7 and/or [TA]8, in the UGT1A1 promoter.
In another embodiment, the present invention provides methods for predicting an individual""s sensitivity to xenobiotics by screening individuals for variation in glucuronidation activity by detecting polymorphisms in a UGT promoter and determining the number of TA repeats in that UGT promoter. More specifically, the present invention provides methods for predicting an individual""s sensitivity to xenobiotics based upon varying glucuronidation activity caused by the presence or absence of alleles [TA]5 [TA]6, [TA]7 and/or [TA]8 in the UGT1A1 promoter.
In a preferred embodiment, the method comprises the steps of obtaining a DNA sample from an individual, amplifying the DNA comprising all or part of a UGT1 promoter which comprises the TATA box sequence upstream of the UGT1 exon 1, determining the number of TA repeats in the amplified DNA by gel electrophoresis or sequencing the amplified DNA.
Preferably the DNA is amplified using the polymerase chain reaction (PCR) using a radioactively labeled pair of nucleotide primers that are designed to amplify the region of the promoter known to regulate expression of the UGT gene.
Preferably, the DNA region correlating with expression levels of UGT is the promoter region comprising the TATA repeat sequence in which polymorphisms in the number of TA repeats has been shown to correlate with expression of the UGT gene. More preferably, the polymorphisms comprise [TA]5 and/or [TA]8.
The invention further comprises a kit for screening individuals to detect polymorphisms in a UGT gene promoter, the kit comprising primers for amplifying DNA in a region comprising all or part of a UGT promoter which comprises the TATA box sequence upstream of the UGT exon 1. In a preferred embodiment, the kit also contains deoxynucleoside triphosphates, buffers, labels for detecting the polymorphisms and instructions. Most preferably, the polymorphisms comprise the homozygous genotypes [TA]5/[TA]5, and/or [TA]8/[TA]8, as well as the heterozygous genotypes [TA]5/[TA]6, [TA]5/[TA]7, [TA]5/[TA]8 [TA]6/[TA]8, and [TA]7/[TA]8.
In another embodiment, the invention further comprises a kit for screening individuals for variation in glucuronidation activity by detecting polymorphisms in a UGT gene promoter, the kit comprising primers for amplifying DNA in a region comprising all or part of a UGT promoter which comprises the TATA box sequence upstream of the UGT exon 1. In a preferred embodiment, the kit also contains deoxynucleoside triphosphates, buffers, labels for detecting the polymorphisms and instructions. Most preferably, the polymorphisms comprise the homozygous genotypes [TA]5/[TA]5 and/or [TA]8,/[TA]8, as well as the heterozygous genotypes [TA]5/[TA]6, [TA]5/[TA]7, [TA]5/[TA]8, [TA]6/[TA]8, and [TA]7/[TA]8.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which described presently preferred embodiments thereof and are not intended to limit the invention as described in the claims.